The action of 3 alkylating carcinogens (dimethylnitrosamine, diethylnitrosamine, and methylnitrosourea) on nucleolar and nuclear DNA in normal and regenerating rat liver will be studied. DNA will be isolated at various time points after treatment. Base alkylations and phosphate alkylations will be quantitatively analyzed by enzymatic digestion to deoxyribonucleoside 5'-monophosphates, chemical labeling with a radioactive reagent of high specific activity (see below), separation of the radioactive products by thin-layer chromatography, and counting. The main objective of these studies is to determine whether the alkylation of nucleolar DNA differs from that of nuclear DNA and whether replicating DNA in regenerating liver exhibits an altered susceptibility to alkylating agents compared to DNA of normal adult liver. The initial formation of individual alkylated products and their persistence will be assayed quantitatively. To be able to conduct the quantitative analytical studies on small amounts of nucleolar DNA a radiochemical method will be developed. (32P)- and (125I)-labeled phosphorimidazolidates will be synthesized as chemical phosphorylating reagents capable of conferring label of high specific activity to nucleotides in enzymatic DNA digests, resulting in the formation of radioactive P1,P2-diesterpyrophosphates.